Alpha-1 antitrypsin (A1AT) is the major circulating serine protease inhibitor. Hypersialylated glycoforms (HSG) are produced to boost A1AT anti-inflammatory and anti-protease properties. Their occurrence and prognostic impact outside severe COVID-19 or community-acquired pneumonia are unknown. Our aim was to clarify the occurrence of A1AT functional deficiency and HSG in patients admitted into intensive care unit (ICU) for any cause. A1AT and elastase inhibitory capacity (EIC) were measured in serum. Functional A1AT deficiency was defined by a measured EIC/calculated EIC Ratio ≤0.85. HSG were identified by isoelectrofocusing and quantified by gel densitometry. A total of 248 serum samples was analyzed, 173 from COVID-19 and 75 from non COVID-19 patients. A functional A1AT deficiency occurred 3-fold more frequently in non-COVID-19 than in COVID-19 patients: 18.7 % vs 6.9 % and was not associated with more frequent S/Z deficient alleles. Functional deficiency was also more frequent in deceased than alive patients in COVID-19 group. M0 and M1 HSG of A1AT occurred in around half of patients but the relative proportion of M1 significantly increased in deceased vs alive patients only in the non-COVID-19 group explaining the absence of worsening of the functional deficiency. In conclusion, our study shows that a functional A1AT deficiency is more frequently observed in patients admitted to the ICU for a cause unrelated to COVID-19, as well as in those with an unfavorable evolution. Among the latter, only those admitted for non-COVID-19 tried to compensate the functional deficiency by increasing the proportion of M1 HSG of A1AT.

Journal Article Carfilzomib and Proteinuria: Potential Interference or Iatrogeny? Get access Lucie Vaudran, Lucie Vaudran CHU Lille, Department of Biology Pathology Genetics, Lille, France Address correspondence to this author at: CHU Lille, Department of Biology Pathology Genetics, rue du Pr Jules LECLERCQ, 59037 Lille Cedex, France. E-mail lucie.vaudran@chu-lille.fr. https://orcid.org/0000-0003-0574-7595 Search for other works by this author on: Oxford Academic Google Scholar Fabienne Loiseleur, Fabienne Loiseleur CH Roubaix, Department of Medical Laboratory, Roubaix, France Search for other works by this author on: Oxford Academic Google Scholar Nicolas Nezry, Nicolas Nezry CHU Lille, Department of Biology Pathology Genetics, Lille, France Search for other works by this author on: Oxford Academic Google Scholar Justin Courtin, Justin Courtin CHU Lille, Department of Central Pharmacy, Lille, France Search for other works by this author on: Oxford Academic Google Scholar Susanna Schraen, Susanna Schraen CHU Lille, Department of Biology Pathology Genetics, Lille, France Search for other works by this author on: Oxford Academic Google Scholar Brigitte Onraed Brigitte Onraed CHU Lille, Department of Biology Pathology Genetics, Lille, France Search for other works by this author on: Oxford Academic Google Scholar The Journal of Applied Laboratory Medicine, jfae040, https://doi.org/10.1093/jalm/jfae040 Published: 29 May 2024 Article history Received: 22 January 2024 Accepted: 12 April 2024 Published: 29 May 2024

INTRODUCTION:Total alkaline phosphatase (tALP) levels rise physiologically in maternal serum during pregnancy, and excessively so in certain conditions. However, current reference values are dated, nonlinear, and based on small samples. Factors related to variation in tALP remain unexplained. Thus, our goals in this study were to establish a physiological development curve for tALP within low-risk pregnancies and to evaluate the factors influencing tALP values.METHODS:This was a single-center, retrospective, observational study. All patients who delivered a live singleton infant at our center from January 1, 2011 to May 31, 2019, and had a tALP assay during pregnancy, were included regardless of the gestational age at which the assay was conducted.RESULTS:A total of 2415 pregnancies were included. Median tALP decreased during the first trimester, it increased slightly during the second trimester, and then increased sharply during the third trimester. Factors associated with a significant increase in tALP were chronic histiocytic intervillositis, cholestasis, multiple pregnancies, liver disease, preeclampsia, smoking, and low weight for gestational age. Conversely, gestational diabetes was associated with a discrete decrease in tALP.DISCUSSION:Our large sample allowed establishment of tALP reference curves based on gestational age. To interpret these results more thoroughly, factors that influence tALP rates should be further scrutinized.

L’électrophorèse des protéines (EPS) est une aide au diagnostic et/ou au suivi de pathologies inflammatoires, hépatiques ou hématologiques.

Patients infectedwithSARS-CoV-2maydevelop eithermild or suddenly severe symptoms of acute respiratory infection for yet unknown reasons. However, risk factors of severe Coronavirus Disease-19 (COVID-19) such as age, cardiomyopathy, diabetes, hypertension, obesity and male sex have been identified. In severe cases, the worsening of hypoxemia, characteristic of acute respiratory distress syndrome (ARDS), requires an admission in an intensive care unit (ICU) for ventilatory support [1]. Mortality in these patients is high, around 20–35% depending on series [1]. Post-mortem histopathological analysis of lungs from patients who died from COVID-19 showed bilateral diffuse alveolar damage consistent with an ARDS [2]. Other associated features frequently reported are intra-alveolar fibrin deposition and a strong fibrotic reaction [3], protein-enriched interstitial edema and inflammatory infiltrates dominated by lymphocytes [3] or by pro-inflammatorymacrophages [2]. In several animal models of SARS-CoV-2 infectionhistological analysis of the lungsalso demonstrated a progressive infiltration of alveolar cavities by inflammatory cells mainly T lymphocytes, macrophages but also neutrophils [4]. Regarding pathogenesis,wehypothesize that the release by neutrophils and/or alveolar macrophages of stored proteases such as neutrophil elastase (NE), matrix metalloprotease-12 (MMP-12) or both, contributes to the diffuse alveolar damage. Indeed these proteases are able to degrade elastin fibers [5] which are core components of the interstitial matrix in the airways and fundamental for their viscoelastic properties. The aimof our studywas to determine whether severe COVID-19 patients with different outcomes have different pattern of circulating proteases (NE, MMP-12) and of the major antiprotease alpha-1 antitrypsin (A1AT) during their stay in ICU. Thirty-nine patients admitted into the ICU of CHU Lille for intubation and mechanical ventilation following COVID-19 between 18March and 5May 2020were included. This study is nested within the LICORNE clinical and biological database which received an Ethics Committee approval (CPP Nord-Ouest 4, ECH20/09). SARS-CoV-2 infection was confirmed by RT-qPCR analysis of nasopharyngeal swabs. Patients were followed up longitudinally during the ICU stay. Through reviewing of the electronicmedical records patients were classified into two groups: patients who died during (n=10) vs. patients alive at the end of ICU stay (n=29). Initial blood samples for routine inflammatory markers testing (C-reactive protein [CRP], haptoglobin, transthyretin) were drawn at Malika Balduyck and Pascal Pigny share joint senior authorship.

SCOPE:Type 2 diabetes (T2D) induces organ damage associated with glycation, among other metabolic pathways. While therapeutic strategies have been tested to reduce the formation and impact of glycation products, results remain equivocal. Anti-diabetic therapies using probiotics have been proposed, but their effect upon glycation has not been reported. Here, the effects of the bacterial strain Lactobacillus fermentum ME-3 on glycation and T2D-related complications in a mouse model of T2D are investigated.METHODS & RESULTS:Wild-type LepRdb/+ and diabetic LepRdb/db littermates receive a daily gavage of either water or the probiotic ME-3 strain (1010 CFU). Glycation markers, fructoselysine-derived furosine (FL-furosine) and carboxymethyllysine (CML), are quantified in four major organs and plasma using stable-isotope dilution LC-MS/MS. After 12 weeks of ME-3 treatment, diabetic mice gain less weight and exhibit an apparently improved glucose tolerance. The ME-3 treatment reduces median renal levels of FL-furosine in both genotypes by 12-15%, and renal and pulmonary free-CML in diabetic mice by 30% and 18%, respectively. Attenuated hepatic steatosis and an improved plasma lipid profile are also observed with treatment in both genotypes, while the gut microbiota profile is unchanged.CONCLUSION:L. fermentum ME-3 has therapeutic potential for reducing the formation/accumulation of some glycation products in kidneys and attenuating some common diabetes-related complications.

A 4-year-old boy was evaluated because of confusing urine protein results. No protein was detected by dipstick, but the concentration was 130 mg/dL by a quantitative benzethonium chloride assay. Clinical examination, serum electrolytes, renal function, and renal imaging were normal. Urine immunofixation (Figure 1A) only showed traces of albumin, excluding tubular and glomerular proteinuria, findings confirmed by normal values for urine albumin and β2 microglobulin. The stored urine was gray and turned …

To the Editor: The role of serum free light chains (SFLC) assessment is increasingly important in the management of patients with multiple myeloma (MM), as well as other monoclonal gammopathies. For instance, about 15% of MM secretes light chain only and must be followed using SFLC assays. In other cases, a free light chain escape is observed at relapse. Moreover, the new IMWG criteria also include the ratio of SFLC to define symptomatic MM and to evaluate the response (stringent complete remission). Nonetheless, FLC measurements remain challenging is some cases especially in the context of antigen excess.1 In this case report, we emphasize that this issue can be addressed by using a novel specific SLFC assay. Antigens at high concentrations can interfere with the antigen-antibody reaction and lead to false low SFLC results in patients with MM. Underestimation of SFLC could lead to inappropriate disease management. Some studies have tried to quantify this phenomenon and reported a frequency between 0.12% and 5.4%.1, 2 The immunoturbidimetry Freelite assay (The Binding Site, Birmingham, UK) includes a control step for antigen excess in order to prevent SLFC underestimation. The assay can detect atypical reaction kinetic by measuring the rate of the reaction at three different time windows in order to identify samples that have a relatively high initial rate of reaction. Suspects' samples are re-assayed at the higher re-dilution.2 However, this automated detection of antigen excess remains uncompleted and prone to error. In addition to systematic dilutions difficult to perform routinely on all samples, few solutions exist to solve this pitfall but not prevent it.3 Recently, a new sandwich ELISA assay was developed by Sebia (Lisses, France) can avoid this problem. Indeed, the higher sensitivity of the test allows to use larger dilutions than Freelite (100 times) so that the risk of antigen excess is significantly reduced. Here, we report three cases of antigen excess pitfall with Freelite assay, resolved by Sebia FLC. All data were retrieved from a human biological database authorized by the French Ministry of Research (No.DC-2008-642). Therefore, no written informed consent was collected from this population. The first patient (P1) is a 55-year-old male with asthenia and renal failure. Serum protein electrophoresis (SPEP) and immunofixation (Sebia Hydrasys 2scan) revealed a monoclonal peak in the beta1 globulins area and showed an excess of lambda light chains in addition to a monoclonal IgA lambda. The second patient (P2) is a 74-year-old male with similar symptoms. SPEP revealed two monoclonal peaks in gamma globulins area. The immunofixation test revealed two bands with the anti-lambda antiserum without similar bands with anti-gamma, anti-mu, and anti-alpha antisera. Samples were sent to our laboratory in order to rule out monoclonal IgD or IgE gammopathies. The third patient (P3) is a 62-year-old male with a 10-year history of IgA MM. He was treated with autologous stem-cell transplantation and VTD (bortezomib, thalidomide, and dexamethasone). Second-line treatment consisted of 66-cycles carfilzomib-lenalidomide-dexamethasone. Third-line was pomalidomide-dexamethasone, fourth-line by daratumumab-bortezomib-dexamethasone, and fifth line with doxorubicin-ixazomib-dexamethasone. Finally, he was treated with chimeric antigen receptor T (CAR-T) cells in September 2018. The SPEP and immunofixation revealed a monoclonal peak in the gamma globulins area and showed a monoclonal IgA kappa. A free light chain assay was performed according to the two methods: immunoturbidimetry Freelite assay on the SPAplus analyzer at the recommended initial dilution of 1/10 and ELISA assay Sebia FLC on the das AP22 ELITE at the recommended initial dilution of 1/1000. Lambda free light chains concentrations with Freelite assay were moderately increased with 131 mg/L for P1 and 159 mg/L for P2 at initial dilution (Table 1). Kappa free light chains concentration with Freelite assay was lower than 4.1 mg/L for P3. The serum involved/uninvolved free light chain ratios (iFLC/uFLC) were lower than 100. The analyzer SPA Plus did not report any alarm for antigen excess. Given the results of serum immunofixation, additional manual dilutions were performed at 1/1000 and 1/10 000, providing very high concentrations of involved free light chains: 29 238 mg/L for P1, 57 922 mg/L for P2, and 2630 mg/L for P3. At the last dilution, iFLC/uFLC ratios were higher than 100. Comparatively, Sebia FLC assay gave values above 90 mg/L for all samples at initial dilution. The final results of involved free light chains after automatic dilutions at 1/100 000 were 1791 mg/L for P1, 1138 mg/L for P2, and 3339 mg/L for P3. Our report illustrates the possible underestimation of Freelite SFLC in case of antigen excess. In these three cases, the new Sebia FLC assay overcame this difficulty without any antigen detection system like Freelite by using an initial dilution 100 times greater combined with multiples wash steps. We noticed significant differences between the final results of the two methods. This is consistent with recent studies reported that Freelite assay provide higher values than Sebia FLC, and the bias increased proportionally with SFLC levels.4 However, differences observed between the two assays in our cases could not completely relate to the negative bias in high values described for the Sebia method. Measurements obtained with Freelite reagent are obviously overestimated and quantitatively inconsistent with the electrophoretic profiles and could be explained by FLC polymerization. Indeed, FLC molecules are usually monomers or dimers5 but higher polymeric forms frequently occur. The polymers act as multiantigenic targets in immunoprecipitation assays, which accelerate aggregates formation and leads to overestimation of antigen measurement.6 These three cases also highlight the importance of a careful analysis of SFLC results, in conjunction with other tests such as SPEP, urine electrophoresis, immunotyping, bone marrow biopsy immunohistochemistry, and clinical history; especially since the phenomenon can even occur for commonly measured SFLC concentrations for patient 3. In conclusion, our report shows that the new sandwich ELISA Sebia FLC is a good alternative to SFLC assessment and can avoid the problem of antigen excess. The results also emphasize the variability of SFLC measurements depending on SFLC assays and related to polymerization. With the development of new tests for SFLC measurement, a standardization of these assays will therefore be necessary. Nothing to report. Pekar: designed and performed the research study, main writer. Grzych and Manier: advised and corrected the manuscript. Schraen and Onraed: designed the research study and corrected the manuscript.

Purpose: Accurate quantification of monoclonal IgM immunoglobulins is essential for response assessment in patients with Waldenström's macroglobulinaemia (WM). The propensity of IgM to form multimers in serum makes sample evaluation by current laboratory methods particularly challenging. Experimental Design: We assessed the precision and linearity of IgMκ and IgMλ heavy/light chain (HLC, Hevylite) assays, and established reference intervals using 120 normal donor sera. We compared the quantitative performance of HLC assays with serum protein electrophoresis (SPE) and total IgM nephelometry for 78 diagnostic samples and follow-up samples from 25 patients with WM. Comparisons were made between the three methods for diagnostic sensitivity and response assessment. Results: IgMκ and IgMλ HLC assays showed low imprecision and good linearity. There was good agreement between summated HLC (IgMκ + IgMλ) and total IgM (measured nephelometrically; R2 = 0.90), but only moderate agreement between involved IgM HLC and SPE densitometry (R2 = 0.49). Analysis of 120 normal donor sera produced the following normal ranges: IgMκ: 0.29–1.82 g/L; IgMλ: 0.17–0.94 g/L; IgMκ/IgMλ ratio: 0.96–2.30. Using these ranges, IgM HLC ratios were abnormal in all WM presentation sera tested, including 15 with non-quantifiable SPE. Despite discordance in quantitation, responses assigned with HLC assays showed excellent agreement to those based on international guidelines using SPE or total IgM; although abnormal HLC ratios indicated residual disease in some patients with negative electrophoresis results. Conclusions: Nephelometric assessment of IgMκ and IgMλ HLC pairs offers a quantitative alternative to traditional laboratory techniques for the measurement of monoclonal IgM and may aid in the management of WM. Clin Cancer Res; 22(20); 5152–8. ©2016 AACR.

Purpose: Accurate quantification of monoclonal IgM immunoglobulins is essential for response assessment in patients with Waldenstr€ om's macroglobulinaemia (WM). The propensity of IgM to form multimers in serum makes sample evaluation by current laboratory methods particularly challenging. Experimental Design: We assessed the precision and linearity of IgMk and IgMl heavy/light chain (HLC, Hevylite) assays, and established reference intervals using 120 normal donor sera. We compared the quantitative performance ofHLCassayswith serum protein electrophoresis (SPE) and total IgM nephelometry for 78 diagnostic samples and follow-up samples from 25 patients with WM. Comparisons were made between the three methods for diagnostic sensitivity and response assessment. Results: IgMk and IgMl HLC assays showed low imprecision and good linearity. There was good agreement between summated HLC (IgMk þ IgMl) and total IgM (measured nephelometrically; R 1⁄4 0.90), but only moderate agreement between involved IgM HLC and SPE densitometry (R 1⁄4 0.49). Analysis of 120 normal donor sera produced the following normal ranges: IgMk: 0.29–1.82 g/L; IgMl: 0.17–0.94 g/L; IgMk/IgMl ratio: 0.96–2.30. Using these ranges, IgM HLC ratios were abnormal in all WM presentation sera tested, including 15 with non-quantifiable SPE. Despite discordance in quantitation, responses assigned with HLC assays showed excellent agreement to those based on international guidelines using SPE or total IgM; although abnormal HLC ratios indicated residual disease in some patients with negative electrophoresis results. Conclusions: Nephelometric assessment of IgMk and IgMl HLC pairs offers a quantitative alternative to traditional laboratory techniques for the measurement of monoclonal IgM and may aid in the management of WM. Clin Cancer Res; 1–7. 2016

The combination of pomalidomide and low-dose dexamethasone (Pom-Dex) can be safely administered to patients with end-stage relapsed/refractory multiple myeloma (RRMM). However, we observed a shorter median progression-free survival (PFS) and overall survival (OS) in these patients when characterized with adverse cytogenetics (deletion 17p and translocation [4;14]) in the Intergroupe Francophone Myélome (IFM) 2009-02 trial. We then sought to determine whether MM with adverse cytogenetics would benefit more from Pom-Dex if exposed earlier in the multicenter IFM 2010-02 trial. The intention-to-treat population included 50 patients, with a median age of 63 years (38% were ≥65 years). Interestingly, there was a striking difference in time to progression (TTP), duration of response, and overall response rate (ORR) according to the presence of del(17p) compared with t(4;14) (TTP, 7.3 vs 2.8 months; duration of response, 8.3 vs 2.4 months; and ORR, 32% vs 15%). OS was prolonged after Pom-Dex, particularly in t(4;14), given the short TTP, suggesting that patients were rescued at relapse with further lines of therapy. Pom-Dex, a doublet immunomodulatory drug-based regimen, is active and well tolerated in adverse cytogenetic patients with early RRMM, particularly in those with del(17p), who are characterized by a high and rapid development of a refractoriness state and known for their poor prognosis. Future studies will determine the underlying mechanisms of Pom-Dex activity in del(17p). This trial is registered at www.clinicaltrials.gov as #NCT01745640.

Abstract Background Quantification of monoclonal immunoglobulins in serum is the foundation of IMWG multiple myeloma (MM) response criteria for patients with intact immunoglobulin disease. However, electrophoretic methods for quantifying M-Ig in serum are subject to well documented limitations including inaccuracy at high concentrations (due to dye saturation) and poor sensitivity at low concentrations. Moreover, the methods require skilled interpretation and can be time consuming; however their clinical utility is well established. Recently heavy/light chain (HLC) assays, which quantify kappa and lambda isotypes of intact immunoglobulins have become available. Here we compare responses assigned by the immunoassays to IMWG criteria and evaluate the clinical impact of discordance. Methods Sequential sera, from enrolment to progression, were available for 107 MM patients (59 IgGκ, 29 IgGλ, 12 IgAκ, 7 IgAλ) enrolled onto either the IFM 2009-02 end-stage relapsed or refractory MM or the IFM 2010-02 in del17p and t(4;14) relapsed or refractory MM trials. The inclusion criterion was a measurable intact immunoglobulin MM according to IMWG criteria (M spike ≥ 10 g/L), using serum and/or urine protein electrophoresis. IgA HLC (IgAκ and IgAλ) and IgG HLC (IgGκ and IgGλ) analysis was compared to historic SPEP, IFE, UPEP, uIFE and serum free light chain (sFLC) results (measured using polyclonal antisera based assays). Responses were assigned at approximately 90 days (median 90, range 61-107 days) and at maximum response (if different) according to IMWG criteria using changes in monoclonal protein concentrations measured either by SPEP or dHLC (clonal - non clonal). Complete response was assigned either by the absence of monoclonal protein on IFE or by a normal HLC ratio. Results At the time of enrolment, 87/88 IgG and 17/17 IgA patients had abnormal HLC ratios which were concordant with IFE results, and quantification of the monoclonal protein by dHLC was similar (median (range): 29.2 (2.5-77.5) g/L) and SPEP (30.7 (1.8-66.9) g/L). After 3 months of treatment, responses assigned by HLC/FLC assays showed near-perfect agreement with responses assigned using SPEP and IFE (Weighted Kappa >0.81, p<0.001). Analysing patients with ≥ partial response (PR) or At maximum response there was near-perfect agreement between the responses assigned using HLC/FLC or SPEP, IFE, UPEP, uIFE (Weighted Kappa >0.81, p<0.001). Changes in dHLC predicted response in 16/39 patients (15 IgG and 1 IgA), achieving ≥PR up to 224 days earlier than SPEP (median 52 days, range 22-224 days). In addition, in 10/79 patients, changes in dHLC indicated relapse up to 77 days (median 56 days range 28-77 days) earlier than standard methods. Conclusion Changes in dHLC can be used in place of SPEP quantification to assign IMWG responses in patients with intact immunoglobulin MM. Minor discordances had an impact on the model fit favouring the HLC assigned response over response assigned using SPE/IFE. Changes in dHLC identified response and relapse earlier than clinically assigned in a number of patients. Considering the small number of patients, we suggest that larger prospective trials be conducted to validate these findings. Disclosures Leblond: GSK: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau. Snell:The Binding Site Group Ltd: Research Funding. Leleu:Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; BMS: Honoraria; Amgen: Honoraria.

Background. Waldenstrom’s macroglobulinaemia (WM) is a B cell lymphoproliferative disorder which is characterised by the production of monoclonal IgM (m-IgM). Response assessment is reliant upon reductions in the m-IgM as measured by serum protein electrophoresis (SPEP) or nephelometric/turbidimetric total IgM (tIgM) measurements alongside lymphadenopathy / splenomegaly assessment. Response assessment guidelines include reductions of <25% m-IgM (stable disease, SD), 25-50% (minor response, MR), 50-90% (partial response, PR), >90% (very good partial response, VGPR), 100% with negative immunofixation (complete response, CR) with progressive disease being described as a >25% increase in m-IgM. However, quantitation of m-IgM is not without limitations. The pentameric form of IgM has a molecular weight of >900 kDa, which may be responsible for its tendency to self-aggregate which causes issues in the accuracy of quantitation by SPE. tIgM values are systematically greater than measurement by SPEP densitometry and unable to discriminate between m-IgM and polyclonal IgM as serum concentrations approach the normal range (~2 g/L). Recently novel immunoassays quantifying the heavy / light chain partnership for IgM (IgMκ / IgMλ) have become available, here we report on the performance of the assay in a population of Waldenstrom’s patients.

Bing-Neel syndrome (BNS), a rare neurological syndrome associated with Waldenström macroglobulinaemia (WM), is a direct involvement of the central nervous system by lymphoplasmacytoid cells characterized with an adverse prognostic. The MYD88 L265P mutation has been identified in the vast majority of patients with WM. The diagnosis of BNS is often challenging because of the variety of clinical presentations associated with difficult histological techniques. We hypothesized that identification of MYD88 L265P mutation in the cerebrospinal fluid (CSF) would contribute to the diagnosis of BNS in addition to imaging, flow cytometry and cytology. We identified MYD88 L265P mutation in the CSF and the bone marrow of all cases of BNS using quantitative polymerase chain reaction qPCR and Sanger sequencing. Copy neutral loss of heterozygosity including MYD88 was observed in one case. No mutation of CXCR4, CD79A and CD79B was observed in parallel. We further showed that monitoring the quantitative expression of MYD88 L265P mutation might be a useful molecular tool to monitor response to chemotherapy using qPCR. In conclusion, identification of MYD88 L265P mutation might be a new molecular-based biomarker tool to add to the diagnostic and monitoring armamentarium for BNS.

Background. The depth of Hypogammaglobulinemia has been related to adverse prognosis in myeloma for decades, but most importantly, it has been suggested that its recovery following treatment was associated with good outcome and prolonged survival. However, none of the traditional techniques has allowed a precise measurement of isotype-matched (i.e. concentrations of IgGκ in an IgGλ myeloma patient) hypogammaglobulinemia. Recently, a new test quantifying paired clonal and non-clonal immunoglobulins (heavy/light chains HLC i.e. IgGκ/IgGλ) in serum was developed. Here we aim to assess the new HLC assays as tools to measure Hypogammaglobulinemia, and potentially replace traditional techniques for the monitoring of patients with myeloma.

Background. Protein electrophoresis and immunofixation in the serum (SPEP - SIF) and urine (UPEP – UIF) have been routinely used for decades for characterizing and quantifying the M protein in Multiple Myeloma (MM). However, these techniques are notoriously tarnished with inaccuracy, despite improvements in recent years. The most important breakthrough in the field in recent years was the discovery of the Serum Free Light Chain Assay (sFLC), a routine quantitative and automated assay that measures kappa and lambda sFLC, however this was added to / rather than replaced traditional tests in the diagnostic armamentarium of MM. Recently, a new test quantifying paired clonal and non-clonal immunoglobulins (heavy/light chains HLC i.e. IgGκ/IgGλ) in serum was developed. Here we aim to assess the new HLC assays as tools to replace SPEP / IFE during MM patient monitoring

Abstract Abstract 3970 Background: Measurement of serum M-spike is used to assess response to therapy and treatment-free survival in IgA myeloma. However, its resolution on SPEP and the presence of IgA-dymers can make accurate measurement difficult. The more the M-spike decreases on SPEP, the more imprecise the M-spike measurement is using SPEP. IgA M-spike often migrates in the betaglobines that renders the M-spike measurement often complicated to analyze. Furthermore, quantification of the clonal IgA chain by nephelometry (IgAneph), which inherently includes monoclonal and polyclonal immunoglobulins, does not accurately reflect the tumour burden. Currently, there is a need in myeloma with IgA isotype (approximately 30–40% of patients) to identify new markers that better reflect the disease burden and the response to treatment, and correlate to patients' outcome. Hevylite® measures IgAkappa and IgAlambda and might provide precise quantitative measurement of the IgA M-spike. We sought to determine whether Hevylite® can be used as a reliable marker for diagnosis and response to therapy in IgA myeloma as compared to the M-spike measurement on SPEP and nephelometry. Methods: We conducted a retrospective analysis on 113, smoldering or symptomatic, IgA myeloma patients at diagnosis referred to our department from 1997 to 2011. All serum samples were collected prior to treatment or at relapse (for sequential data) and were kept frozen since collection. Hevylite® measurements were made at The Binding Site Ltd, Birmingham, UK. A normal range was produced from normal (blood donor) sera (n=138), and were for IgA kappa 0.48–2.82g/L, IgA lambda 0.36–1.98g/L and IgA K/L ratio 0.80–2.04. For ease of comparison we have studied the IgA hevylite ratios expressed as IgA K/L ratio. Results: The median age at diagnosis was 65 years (range: 33–93) and the M/F sex ratio was 0,91. Forty-eight percent of patients had an ISS greater than 2 (n=86). On nephelometry the median IgA level was 22g/L (range min-max, 0–100). Fifty patients had M-spikes migrating among the betablobulines, 27 among the gammaglobulines, 10 migrated in both and 23 unspecified. Fourteen patients had oligosecretory disease (M-component < 10g/L). Forty-two patients had an IgA kappa clonal chain, 70 others an IgA lambda and for one patient the data was not available. Among IgA kappa patients the mean HCLratio was 616.6 (median 117.7 [0.021–4323.7]) whereas it was 0.61 (0.21 [0.004–0.455]) among the IgA lambda patients. Across the entire population, 58 patients were identified by SPEP and HCL ratios. Among the 55 patients whose M-component was not quantified on SPEP, HCL ratios were abnormal in 53/55 (96%) cases. In the subgroup of patients whose M-protein migrates in the beta-region 29 out of 50 are identified by HCL ratios and SPEP. Another 19 patients (38%) had abnormal HCL ratios while unquantified on SPEP. The same was seen in patients whose M-protein migrates among the gammaglobulins with 16/27 and 11/27 (40%), respectively. In the last subgroup of patients with beta and gamma migrating M-proteins, all (10/10) were identified by HLC and SPEP. More interestingly, among the oligosecretory MM patients identified by an M-component < 10g/L, all (14 patients) had an abnormal HCL ratio. In our series of IgA myeloma, 51% of patients were accurately quantified on SPEP. When using HCLratios, an extra 47% of patients became measurable, with an abnormal HLC ratios allowing IgA myeloma in up to 98% of cases. When considering IgA measurement using nephelometry, 102 patients had both high IgA levels (90%) and abnormal HCL ratios. Interestingly, 7 patients had normal IgA levels with IgAneph and abnormal HCL ratio offering a useful diagnostic tool for 96% of patients as compared to IgAneph. In our series, IgA myeloma was neither associated with a poor outcome (median [range] OS: 119 months [0,5–604]) nor a poor response to therapy (median [range] TTP: 16 months (1–92). HCL involved chain ROC analysis identified a 37g/L cut-off as prognostic in IgA myeloma(p=0.039). Conclusion: Hevylite® is a new and reliable marker for diagnostic and monitoring of IgA myeloma. It enables to quantify accurately up to 98% of IgA-MM patients. These preliminary data need confirmation in further prospective trials in order to monitor further the impact of this marker in IgA myeloma patients before it becomes the gold standard to monitor the IgA M-protein in years to come. Disclosures: Boyle: Chugai: Consultancy, Honoraria. Combat:The Binding Site: Employment. Pietrantuono:The Binding Site: Employment. Facon:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees. Harding:The Binging Site: Employment. Leleu:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Sanofi: Honoraria; Onyx: Honoraria, Speakers Bureau; LeoPharma: Honoraria, Speakers Bureau.

Background: The Prognostic Inflammatory and Nutritional Index (PINI) is a simple scoring system that aggregates two blood markers of inflammatory [C-reactive protein (CRP) and orosomucoid] and of nutritional (albumin and prealbumin) states. It is used in routine practice in geriatric medicine, especially in hospitalized elderly patients. This study was undertaken to evaluate the usefulness of PINI index in multiple myeloma (MM), a malignancy of the elderly. Method: The PINI score was determined in 231 previously untreated patients with MM, of whom 112 were 65 yrs old. The serum albumin, prealbumin, orosomucoid (human a1-acid glycoprotein), and hsCRP are measured routinely by immunonephelometry. Results: In the overall population and the elderly subset, PINI 4 (` high PINI') was correlated with a shorter median survival, 26 vs. 65 months in the high and low PINI groups, respectively. The prognostic impact of PINI index was dramatic in the elderly MM subgroup, 6 and 45 months, respectively. The high PINI index also predicted for shorter survival in various groups with good prognostic, such as low International Staging System (ISS) stages, low b2m, and absence of del17p and t(4; 14), further demonstrating its prognostic impact on overall survival. In multivariate analysis, PINI index provided additional survival prognostic information to b2m in a b2m/PINI model. Conclusion: PINI index appears to be a useful and easy-to-perform marker in routine to determine the prognosis of patients with MM, especially in the elderly population. PINI might represent an alternative to ISS score, especially in elderly patients, in the future.

Abstract Abstract 5076 Background. Waldenstrom macroglobulinemia (WM) is a low grade B cell lymphoma characterized by bone marrow infiltration of lymphoplasmacytic tumor cells that secrete monoclonal IgM (M-protein) into the serum. Measurement of the serum M-protein [using serum protein electrophoresis (SPEP)] and measurement of total IgM (using nephelometry) are used to diagnose and monitor WM. There are, however, many limitations with these techniques and new markers are needed. IgG and IgA Hevylite® immunoassays have been reported to be more sensitive than SPEP and nephelometry for identifying monoclonal immunoglobulins in multiple myeloma and unlike immunofixation, provide quantitative information. We hypothesized that serum IgM Hevylite assays (specifically measuring IgMkappa and IgMlambda, separately) would accurately identify serum IgM M-proteins. We also evaluated the association between known tumor burden markers and prognostic factors with IgM Hevylite results in patients with WM. Method. We retrospectively measured IgMkappa and IgMlambda in sera from 59 WM patients: 44 patients were at diagnosis and 15 had relapsed disease. The diagnosis of WM was made according to the current guidelines. All serum samples were kept frozen in the Lille serum bank since collection. All patients gave informed consent prior to the collection and none were treated at time of collection of the serum. Approval of this protocol was obtained from the CHRU Lille and was in accordance with the Declaration of Helsinki. Hevylite measurements were made at The Binding Site Ltd, Birmingham, UK. A normal range was produced from normal (blood donor) sera (n=120), median (and 95%ile ranges) were; IgMkappa 0.634g/L (0.29-1.82), IgMlambda 0.42g/L (0.17-0.94), IgMkappa/IgMlambda ratio 1.6 (0.95-2.3). For ease of comparison IgM hevylite ratios were expressed as the involved monoclonal immunoglobulin/uninvolved polyclonal immunoglobulin (IgMi). To describe the distribution of IgMi Hevylite levels in patients with WM, the median and range (min-max) were reported. Median values were compared using the Wilcoxon rank-sum test and ANOVA. Fisher's exact test was used to compare proportions. All statistical tests were two-sided. All analyses were conducted using SPSSv12 software. Results. The baseline characteristics of the patients were as follows: the median (range) age was 68 years (41-86), male/female 38/21, serum b2M 3.0mg/L (1.2-9.0), hemoglobin 11.8g/dL (7.6-15.4), platelet count 267 ×109/mm3 (55-741), serum M-spike 19g/L (3.0-52.7). In our series, 18 (31%), 22 (37%) and 19 (32%) patients had low, intermediate and high risk disease respectively, in the WM-IPSS scoring system. The median (min-max) IgMkappa ratio was 134 (8.7-2850) and IgMlambda ratio was 0.03 (0.0007-0.39). IgMi Hevylite ratio was 98.07 (2.59-2850). The IgMi Hevylite correlated well with the M-spike measured using SPEP (r=0.601, p<0.0001). In our study, high IgMi Hevylite levels correlated well with markers of high tumor burden and of poor prognosis. The median (range) IgMi Hevylite level was higher in patients with hemoglobin <10g/dL versus ≥10 g/dL, 267 (8.1-1722) and 76 (2.6-2850) respectively (p=0.013). The median (range) IgMi Hevylite ratio level was significantly (p=0.033) higher in the high risk IPSS group 208 (2.6-2850) than in the intermediate 75 (15-1033) and low risk groups, 98 (8-571). The IgMi Hevylite levels also separated WM patients with progressive disease who required therapy. Twenty seven pts were symptomatic and required specific treatment for WM, and 32 pts were left untreated. The median IgMi Hevylite ratio was significantly higher in progressing patients, 210 (8.1-2850) and 60 (2.6-571) in the 2 groups, respectively (p=0.014). Conclusion. In this study we demonstrated that IgM Hevylite measurement is a new and reliable marker for monitoring WM disease. It is related to poor prognostic markers that separate WM patients with progressive disease who require therapy. We are currently expanding the cohort to confirm these observations. These findings have implications in the management of patients with WM. Disclosures: Leblond: Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Mundipharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genzyme: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Leleu:Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Abstract Abstract 2493 Poster Board II-470 Background. The Prognostic Inflammatory and Nutritional Index (PINI) is a simple scoring system which aggregates two blood markers of inflammatory (C reactive protein and orosomucoid) and of nutritional (albumin and prealbumin) states. This marker is predictive of nearest lethality and chronic institutionalization in geriatric routine practice for hospitalized elderly patients. MM is a malignant plasma cell disorder with a median age around 70 years old at diagnosis, a population characterized with several comorbid conditions that indirectly determine the prognosis and the treatment decision. The current most powerful adverse prognostic markers in MM are the adverse karyotypic abnormalities, such as 17p deletion and (4;14) translocation, and the high serum beta-2 microglobulin (b2m) that corresponds to the high International Staging System (ISS) group in combination with albumin. However, approximately 70% of MM patients have no know adverse karyotypic abnormalities, and high b2m and ISS are essentially informative for a minority of patients. We have sought to evaluate the prognostic value of PINI in MM in a large retrospective study. Method. The PINI score was determined in 226 previously untreated MM patients from sera collected at diagnosis before treatment, of whom 112 pts were 65 or older. The Serum albumin, prealbumin, orosomucoid (human α1-acid glycoprotein) and hsCRP were measured by immunonephelometry. PINI is calculated with the following formula: PINI=[orosomucoid (mg/L) × CRP (mg/L)]/[albumin (g/L) × prealbumin (mg/L)]. To avoid inadequate value of CRP, patients with fever and documented infection at diagnosis were excluded from the study. Results. Main patients characteristics at sample collection for the overall population were as follows: median age=64, ISS 2 and 3 of 29% and 21%, respectively. 35% of pts had chromosome 13 deletion (del13q). These characteristics were similar in pts older than 65. With a median follow-up of 57 months, median (+/-se) overall survival (OS) were 57 (+/-7) and 40 (+/-5) months in the overall and elderly population, respectively. The median (min-max) value of PINI were 0,29 (0,01-123) and 0,47 (0,01-123), respectively. The most informative PINI cut-off was determined at 4 and corresponded to its best impact on survival; 14% and 12.5% of pts had PINI ≥ 4, in the overall and elderly population, respectively. PINI correlated with markers of tumour burden such as high b2m level, low hemoglobin level and, high creatinin value. PINI ≥ 4 was also related to high ISS score (II + III), in 46% and 32% of pts, respectively. PINI did not correlate to del13q. In univariate analysis, median OS (+/-se) was significantly lower in patients with PINI ≥ 4 (number of death/number of pts in the group (O/N)=24/31) than pts with PINI <4 (O/N=99/195), 26 (±12) vs. 65 (±7) months, [p=0.0003; OR=2.21] respectively. This adverse prognostic impact was even more dramatic in elderly patients, 6 (±7) months (O/N=14/112) vs. 45 (±7) months (O/N=60/96), p<0.0001, respectively. Other markers, age above 65, low haemoglobin value below 10g/dL, b2m above 4mg/L, presence of del13q and ISS score II +III remained significant but with a lower impact. Interestingly, PINI ≥ 4 identified a subgroup of patients characterized with intermediate prognostic in pts with low b2m (< 4 mg/L) [PINI ≥ 4: O/N=8/12, 50 (±29) vs. PINI <4: O/N=53/123, 79 (±6), p=0.02], hemoglobin value ≥ 10g/dL [PINI ≥ 4: O/N=9/12, 13 (±10) vs. PINI <4: O/N=54/135, 79 (±8), p=0.0003] and absence of del13q [PINI ≥ 4: O/N=10/15, 50 (±18) vs. PINI <4: O/N=36/86, 80 (±13), p=0.0018]. In multivariate analysis, factors evaluated for association with high PINI were age (>65years), hemoglobin (<10g/dL), platelet count (<100 ×109/L), b2M (≥4mg/L) and ISS (II+III). Modeling results showed a significant association of PINI with elevated b2M and del13q, with 51% (17/33) vs. 72% (16/22) of patients with high PINI in b2M < 4 mg/L vs. ≥ 4 mg/L (OR=2.4, p=0.003), and with 56% (34/60) vs. 85% (6/7) of patients with high PINI in absence vs. presence of del13q (OR=2.3, p=0.001). Conclusion. PINI appeared to be a useful and easy-to-perform marker in routine practice to determine the prognosis of patients with MM, especially in the elderly population with no know adverse karyotypic abnormalities and low b2m/ISS. The study of PINI should be performed in future prospective clinical trials to validate these results. Disclosures: No relevant conflicts of interest to declare.